1. Field of the Invention
The present invention relates to the fields of molecular biology and molecular diagnostics. The present invention provides improved methods and compositions for amplification of nucleic acids. In particular, the invention includes materials and methods for the amplification and/or detection of target nucleic acid sequences by nested PCR.
2. Related Art
PCR amplifies specific nucleic acid sequences through a series of manipulations including denaturation, annealing of oligonucleotide primers, and extension of the primers with DNA polymerase (see U.S. Pat. No. 4,683,202, U.S. Pat. No. 4,683,195, EP 201,184, EP 050,424, EP 084,796, EP 258,017, EP 237,362, U.S. Pat. No. 4,582,788, U.S. Pat. No. 4,683,202, Mullis, K. B. et al. Cold Spring Harbor Symp. Quant. Biol. 51:263 (1986), Saiki, R. et al. Science 230:1350 (1985), Saiki, R. et al. Science 231:487 (1988), and Loh, E. Y. et al. Science 243:217 (1988)). These steps can be repeated many times, potentially resulting in large amplification of the number of copies of the original specific sequence.
Although PCR has the potential to permit enormous amplification of a desired specific sequence, it can also enormously amplify a non-desired sequence for various reasons including mis-annealing of the primers and contamination of the input sample. The contamination problem is particularly severe in laboratories where PCR is heavily used, such as diagnostic laboratories. The basic rule when performing any PCR is that the products generated post-amplification should be kept spatially separated from the targets or reagents to be used in any subsequent PCR reactions. If this is not strictly adhered to, contamination and false positives of all the following PCR reactions are likely to occur. Contamination by the carryover of amplification products from a previous PCR reaction may be due to aerosol formation or contamination of work-surfaces, reagents, pipettes etc. Using separate work areas and sample handling equipment for product handling versus setup and using the enzyme uracil DNA glycosylase (UDG), as described below, can keep this contamination to a minimum.
To prevent carryover from previously amplified PCR reactions in subsequent PCR reactions, several preventive measures have been suggested. One such measure involves substituting dUTP for dTTP in the PCR reaction to generate deoxyuridine containing PCR products. Although the triphosphate form of deoxyuridine, dUTP, is present in living organisms as a metabolic intermediate, it is rarely incorporated into DNA. When dUTP is incorporated into DNA, the resulting deoxyuridine is promptly removed in vivo by normal processes, e.g. processes involving UDG. Both single- and double-stranded DNAs that contain uracil are substrates for UDG. UDG cleaves the N-glycosyl bond between the uracil base and the phosphodiester backbone of DNA. The resulting apyrimidinic DNA molecule becomes susceptible to hydrolysis at high temperatures. Further, the apyrimidinic site in the DNA blocks DNA polymerase from using the DNA strand as a template for the synthesis of a complementary DNA strand (Schaaper, R. et al. Proc. Natl. Acad. Sci. USA 80:487 (1983)). The presence of substantial numbers of apyrimidinic sites in each DNA target molecule interferes with further amplification procedures which use DNA polymerases to amplify target DNA.
To prevent contamination by carryover of reaction products, a sample would be treated with UDG prior to the a PCR reaction to destroy all uracil-containing contaminants. Methods utilizing UDG to prevent carryover and to decrease contamination in standard PCR reactions have been addressed in U.S. Pat. Nos. 5,683,896, 6,287,823, 5,945,313, 5,035,996, 5,229,283, and 5,137,814. All of these methods employ exo-sample nucleotides in combination with a treatment, such as a glycosylase treatment, to overcome the problem of contamination.
In addition to contamination problems, PCR based methodologies also frequently are not sufficiently sensitive to allow target amplification when the target is a rare sequence. The detection of a rare sequence in a sample is limited by the amount of amplified product available to be tested. Rare sequences can be overlooked in conventional analytical practices.
The need for improved sensitivity and specificity in PCR reactions designed to amplify rare sequences is addressed in U.S. Pat. No. 4,683,195, which describes the use of nested primers for increasing sensitivity of single copy genes. According to the method, two pairs of primers are used to amplify first a larger template nucleic acid molecule and, subsequently, a target nucleic acid sequence that is contained in the amplified template molecule. For many research applications (specifically those working with low or poor quality target or rare messages), applying two rounds of PCR markedly enhances the specificity and sensitivity of PCR analysis.
With reference to FIG. 1, classical nested PCR uses two sets or pairs of amplification primers. A first pair of primers-termed the outer primers-are designed to amplify a template nucleic acid molecule as in standard PCR. After amplification of the template, an aliquot from the amplification reaction mixture is typically diluted into a second amplification reaction mixture. The second amplification reaction mixture contains a second set of primers-termed the inner primers-designed to anneal to an internal portion of the template and to amplify a target nucleic acid sequence from the template molecule. The amplification product of the second reaction is, by definition, shorter than the first.
Nested PCR is designed to increase the sensitivity of PCR by directly re-amplifying the product from the outer primer PCR reaction-amplified template-with a second PCR reaction designed to amplify a specific target nucleic acid sequence within the template nucleic acid molecule. Another advantage of nested PCR is its increased specificity, since the inner, nested primers anneal only if the amplified product resulting from the outer primers has the corresponding, specific sequences, i.e., if the proper template has been amplified.
A very distinct PCR product is normally obtained in nested PCR. The profound improvement in amplification efficiency is believed to be attributed to the increased specificity provided by the use of two primer pairs, the large total number of cycles possible and the replenishment of reaction components such as Taq DNA polymerase.
The chance of amplifying non-desired sequences is reduced with nested PCR as compared to regular PCR since non-desired sequences amplified in the first amplification reaction are not likely to contain a sequence to which the primers for the second amplification reaction-the inner primers-will bind. In contrast, performing the same total number of cycles (30 to 40) with either set of primers individually often amplifies non-desired sequences.
A number of variations of nested PCR are known to those skilled in the art. One commonly used variation is semi-nested PCR. In a semi-nested PCR reaction, one inner primer is included in the primary amplification reaction. In certain applications, semi-nested PCR will add enough specificity for a desired targeted PCR product.
Two step reaction protocols for nested PCR have several drawbacks.
First, the presence of the outer primer pair in the second PCR reaction may result in non-specific amplification of undesired sequences or reduced amplification of the target sequence by the inner primers. An additional drawback is the potential for contamination in the samples when the tubes from the first PCR reaction are opened to remove an aliquot to be used for the second PCR reaction.
The possibility of contamination represents a significant problem with classical nested PCR. After completion of the first amplification reaction, the PCR tube must be opened, the amplification reaction mixture diluted, and then an aliquot of the first amplification reaction mixture must be transferred to a second tube for a second amplification reaction. The tube for the second amplification reaction will typically include the components for the second reaction including the inner primers as inner primers are not typically included in the first amplification reaction. The sample handling and the proximity of the amplification product of a first amplification reaction to the reagents used in a second amplification reaction both contribute to an increased risk of contamination of the second amplification reaction. Another disadvantage with this technique is the cost involved in doing two rounds of PCR reactions.
A number of single-tube nested PCR methods have been developed to try to overcome the difficulties associated with classical nested PCR. Some of these methods rely on having the second PCR mixture physically separated from the first reaction mixture, for example, by a mineral oil layer. After the first round of amplification, the two solutions are mixed by centrifugation and then the second PCR reaction begins. Because an oil layer is required, the benefit of using heated-lid thermocyclers, where there normally is no need for an oil layer, is lost. One example of a single-tube, nested PCR protocol that relies on a physical separation of the reaction components is found in U.S. Pat. No. 5,556,773. This patent discloses physically separating the inner primers from the first amplification reaction mixture and, after the first reaction, introducing the inner primers by centrifugation.
U.S. Pat. No. 5,314,809 describes drop-in/drop-out nested PCR, a method that utilizes an inner primer pair having a lower annealing temperature than the outer primer pair resulting in the inner primers not being extended during the first amplification. The drop-in/drop-out one tube nested PCR technique requires that primers be specifically designed to provide inner and outer primer pairs with sufficient differences in annealing temperatures to practice the technique. The inner primers must have a low annealing temperature to prevent them from annealing to the template during the PCR reaction of the outer primer pair. Although, this technique circumvents the addition of an oil layer to separate the reagents of the two separate reactions and the need to mix the two layers after the first reaction, the restrictions imposed on primer annealing temperatures can make it difficult to suitable primer pairs.
Another method that utilizes specially designed primer pairs is disclosed in U.S. Pat. No. 5,340,728. This patent discloses manipulating the primer concentration and annealing times according to a kinetic model in order to optimize amplification of the target nucleic acid sequence.
Each of the methods designed to improve upon nested PCR has its own attendant deficiencies. Some require specialized materials and/or are limited by complicated primer selection criteria and reaction conditions that reduce the general applicability of the methods. There remains a need in the art for simplified methods for increasing both the specificity and sensitivity of nested PCR amplification. Improved methods are desirable that also eliminate processing steps, minimize cross contamination, and subsequent inaccurate results. These and other needs are met by the present invention.